Nena J. Winand

Cytosolic arylamine N-acetyltransferase (NAT) deficiency in the dog and other canids due to an absence of NAT genes

The purpose of this study was to determine the molecular basis in the dog for an unusual and absolute deficiency in the activity of cytosolic N-acetyltransferase (NAT), an enzyme important for the metabolism of arylamine and hydrazine compounds. NAT activity towards two NAT substrates, p-aminobenzoic acid and sulfamethazine, was undetectable in dog liver cytosol, despite substrate concentrations ranging from 10 microM to 4 mM and a wide range of incubation times. Similarly, no protein immunoreactive to NAT antibody was evident on western blot analysis of canine liver cytosol. Southern blot analysis of genomic DNA from a total of twenty-five purebred and mixed bred dogs, and eight wild canids, probed with a full-length human NAT2 cDNA, suggested an absence of NAT sequences in all canids. Polymerase chain reaction amplification of genomic DNA using degenerate primers designed to mammalian NAT1 and NAT2 consensus sequences generated products of the expected size in human, mouse, rabbit, and cat DNA, but no NAT products in any dog or wild canids. These results support the conclusion that cytosolic NAT deficiency in the domestic dog is due to a complete absence of NAT genes, and that this defect is shared by other canids.

Deletion of the dystrophin muscle promoter in feline muscular dystrophy

We have characterized the mutation in a feline model of DMD that selectively eliminates expression of the muscle and Purkinje neuronal dystrophin isoforms. The cortical neuronal isoform was expressed at a detectable level in skeletal muscle in the absence of the muscle promoter and levels of PCR products representing cortical neuronal-type transcripts in dystrophic muscle were comparable to those of normal feline skeletal muscle. Although localized at the sarcolemma, cortical neuronal dystrophin apparently failed to protect skeletal muscle. Neuronal transcripts could not be amplified from feline heart, indicating that these promoters are not active in this tissue in the cat.

Identification of the novel proteins Yip4p and Yip5p as Rab GTPase interacting factors

The Rab GTPases are key regulators of membrane traffic. Yip1p is a membrane protein of unknown function that has been reported to interact with the Rabs Ypt1p and Ypt31p. In this study we identify Yif1p, and two unknown open reading frames, Ygl198p and Ygl161p, which we term Yip4p and Yip5p, as Yip1p‐related sequences. We demonstrate that the Yip1p‐related proteins possess several features: (i) they have a common overall domain topology, (ii) they are capable of biochemical interaction with a variety of Rab proteins in a manner dependent on C‐terminal prenylation, and (iii) they share an ability to physically associate with other members of the YIP1 family.

Mutation in cyclophilin B that causes hyperelastosis cutis in american quarter horse does not affect peptidylprolyl cis-trans isomerase activity but shows altered cyclophilin b-protein interactions and affects collagen folding

Background: Hyperelastosis cutis in horses is caused by a homozygous mutation in cyclophilin B (PPIB). Results: This mutation changes protein-protein interactions of CypB and delays folding of collagen. Conclusion: Mutant CypB has PPIase activity, but the synthesized collagen contains less post-translational modifications of lysine residues. Significance: CypB has other important functions besides its PPIase activity. The formation of these interactions is crucial for a correct biosynthesis of collagen. The rate-limiting step of folding of the collagen triple helix is catalyzed by cyclophilin B (CypB). The G6R mutation in cyclophilin B found in the American Quarter Horse leads to autosomal recessive hyperelastosis cutis, also known as hereditary equine regional dermal asthenia. The mutant protein shows small structural changes in the region of the mutation at the side opposite the catalytic domain of CypB. The peptidylprolyl cis-trans isomerase activity of the mutant CypB is normal when analyzed in vitro. However, the biosynthesis of type I collagen in affected horse fibroblasts shows a delay in folding and secretion and a decrease in hydroxylysine and glucosyl-galactosyl hydroxylysine. This leads to changes in the structure of collagen fibrils in tendon, similar to those observed in P3H1 null mice. In contrast to cyclophilin B null mice, where little 3-hydroxylation was found in type I collagen, 3-hydroxylation of type I collagen in affected horses is normal. The mutation disrupts the interaction of cyclophilin B with the P-domain of calreticulin, with lysyl hydroxylase 1, and probably other proteins, such as the formation of the P3H1·CypB·cartilage-associated protein complex, resulting in less effective catalysis of the rate-limiting step in collagen folding in the rough endoplasmic reticulum.

Cloning, sequencing and expression analysis of the equine hepcidin gene by real-time PCR

Equine serum or plasma iron concentration drops quickly during inflammation. Accumulation of iron inside macrophages and reduction of the intestinal absorption of this element cause hypoferremia during systemic inflammatory processes. These mechanisms are mediated by hepcidin, a 25 amino acids peptide synthesized mainly in the liver in response to iron stores and inflammation. Hepcidin is an important peptide for systemic iron homeostasis and also has antibacterial and antifungal activities. Hepcidin up-regulation is particularly useful during acute inflammation, especially before adaptive immunity occurs, restricting iron availability necessary for pathogenic microorganism growth. Hepcidin gene products have been previously characterized in man, non-human primates, rat, mouse, dog swine, cattle, fishes, reptiles and birds; but until now not in the horse. We have cloned and sequenced equine hepcidin mRNA and performed hepcidin expression analysis in different tissues collected from four healthy horses. The deduced precursor of equine hepcidin was most homologous to Bos taurus and Sus scrofa. The expressed profile of equine hepcidin in liver was very high. Expression in cervical spinal cord and cerebral cortex was much lower than liver but higher than lung, duodenum, stomach, spleen, kidney, skeletal muscle and bladder. This sequence will be helpful for additional studies on iron metabolism and inflammatory process in horses.

Identification, characterization and expression analysis of hepcidin gene in sheep

Hepcidin is part of the innate immune system, and it plays a central role in the regulation of iron homeostasis. This peptide has been previously characterized in man, non-human primates, rat, mouse, dog, swine, cattle, horse, fishes, reptiles and birds but until now not in sheep. The aim of this study was to sequence, characterize and perform hepcidin expression analysis in different tissues collected from healthy sheep. The resulting open reading frame consisted of 249 bp predicted to encode an 82 aa peptide with a putative 23 aa signal peptide, a 34 aa pro-region and the 25 aa mature hepcidin. The deduced sequence of the sheep hepcidin precursor was most homologous to Bos taurus and Bubalus bubalis. Hepcidin was predominantly expressed in liver, although high expression was present in abomasum and lower level expression occurred in other tissues. These findings extend our comparative knowledge showing the relationship of sheep hepcidin to other mammalian hepcidins and will be helpful for additional studies on iron metabolism and inflammatory processes in sheep.

Lipopolysaccharide infusion up-regulates hepcidin mRNA expression in equine liver

Hepcidin has been found to be the key regulator of iron metabolism that leads to hypoferremia during inflammation. Recent work has shown that equine hepcidin is predominantly expressed in the liver of horses. In this study, hepcidin gene expression was determined in the liver and bone marrow of six healthy horses after iv infusion of Escherichia coli O55:B5 LPS. The IL-6 gene expression was also determined in liver and bone marrow samples. Clinical and laboratory evaluations were measured at multiple time points between 0 and 240 h post-LPS infusion (PI). Liver and bone marrow biopsies were taken immediately before (baseline) and at 6 and 18 h PI. In response to endotoxin infusion, all horses showed characteristic clinical signs of endotoxemia. Plasma iron concentration was decreased significantly from the pre-infusion level at 8 h PI. Hypoferremia peak was observed at 12 h and returned to normal levels at 30 h PI. Relative real-time RT-PCR analysis showed that liver hepcidin and IL-6 mRNA expression was up-regulated at 6 h PI. Bone marrow hepcidin relative expression was not influenced by LPS infusion. In another experiment, equine monocyte cultures were stimulated with LPS (1 µg/ml). Monocyte hepcidin and IL-6 gene expression was significantly induced after 2 h of LPS stimulus and returned to baseline levels thereafter. The present study describes that, in horses, LPS infusion up-regulates hepatic hepcidin mRNA expression resulting in early observed hypoferremia and suggests that hepcidin may act as an acute-phase protein in horses.

Allele frequency of hereditary equine regional dermal asthenia in American Quarter horses in Brazil determined by quantitative real-time PCR with high resolution melting analysis.

Hereditary equine regional dermal asthenia (HERDA) is a genetic disorder that occurs in the American Quarter horse (AQH) and is caused by a c.115G>A missense mutation in the peptidylprolyl isomerase B (PPIB) gene. Using a quantitative real-time PCR high resolution melting analysis genotyping assay for the PPIB mutation, the estimated HERDA allele and carrier frequencies in a sample of Brazilian AQHs were 2.9% and 5.8%, respectively.

Influence of experimental inflammatory response on hepatic hepcidin gene expression and plasma iron concentration in sheep

Hepcidin is a highly conserved disulfide-bonded peptide that plays a central role in iron homeostasis. During systemic inflammation, hepcidin up-regulation is responsible for hypoferremia. This study aimed to analyze the influence of the inflammatory process induced by complete Freunds adjuvant (CFA) or lipopolysaccharide (LPS) on the liver expression of hepcidin mRNA transcripts and plasma iron concentration of sheep. The expression levels of hepcidin transcripts were up-regulated after CFA or LPS. Hypoferremic response was observed at 12 h (15.46 ± 6.05 μmol/L) or 6h (14.59 ± 4.38μmol/L) and iron reached its lowest level at 96 h (3.08 ± 1.18 μmol/L) or 16h (4.06 ± 1.58 μmol/L) after CFA administration or LPS infusion, respectively. This study demonstrated that the iron regulatory hormone hepcidin was up-regulated in sheep liver in response to systemic inflammation. These findings extend our knowledge on the relationship between the systemic inflammatory response, hepcidin and iron, and provide a starting point for additional studies on iron metabolism and the inflammatory process in sheep.

Freund's adjuvant-induced inflammation: clinical findings and its effect on hepcidin mRNA expression in horses

Hypoferremia observed during systemic inflammatory disorders is regulated by hepcidin. Hepcidin up-regulation is particularly important during acute inflammation, as it restricts the availability of iron, which is necessary for pathogenic microorganism growth before adaptive immunity occurs. The aim of this study was to evaluate the clinical findings and hepatic hepcidin mRNA expression in horses using a Freunds complete adjuvant (FCA) model of inflammation. The expression of hepcidin mRNA in the liver was determined in healthy horses following two intramuscular injections of FCA at 0 h and 12 h. Plasma iron and fibrinogen concentrations were measured at multiple time points between 0 h and 240 h post-FCA injection (PI). Hepcidin mRNA expression was determined by RT-qPCR using liver biopsy samples performed at 0 h (control), 6 h and 18 h PI. The mean plasma fibrinogen level was significantly different from the control values only between 120 and 216 h PI. The mean plasma iron level was significantly lower than the control between 16 and 72 h PI, reaching the lowest levels at 30 h PI (33 % of the initial value), and returned to the reference value from 96 h PI to the end of the experiment. Hepcidin mRNA expression increased at 6 h PI and remained high at 18 h PI. The iron plasma concentration was an earlier indicator of inflammatory processes in horses when compared with fibrinogen and might be useful for the early detection of inflammation in the horse. FCA administration caused the rapid onset of hypoferremia, and this effect was likely the result of up-regulated hepatic hepcidin gene expression. This study emphasizes the importance of hepcidin and iron metabolism during inflammation in horses.